Function of transfected globin promoters and the globin locus activator in K562 erythroleukemia cells.
We have examined the importance of cis-acting regulatory elements within the human gamma-globin gene promoter and the globin locus activating region in K562 cells. A gamma-globin or beta-globin promoter fragments were fused with the neomycin phosphotransferase gene in a plasmid-based vector (gamma-neo or beta-neo) and transiently transfected by electroporation into K562 cells. Correctly initiated gamma-neo or beta-neo transcripts were detected with an S1 nuclease protection assay that was internally controlled for transfection efficiency and RNA content. We first optimized the conditions for electroporation and then determined that a gamma-globin promoter fragment extending from -299 and +36 was active in the assay but that a beta-globin promoter extending from -375 to +46 was inactive. Deletion of the gamma-globin promoter to -199 did not affect promoter function, but deletion to -160 reduced promoter strength to 70% of that of control. Additional deletion to position -130 reduced promoter strength to 19% of the control value, and to position -61, 8.7% of the control value. Three gamma-globin promoters containing mutations associated with hereditary persistence of fetal hemoglobin (HPFH), -202 C----G, -196 C----T and -117 G----A, were not overexpressed in K562 cells, consistent with the hypothesis that these promoters are not overexpressed in fetal erythroblasts, only in adult red cells. When the beta-globin locus activating region (LAR) was added to a wild-type or an HPFH gamma-neo plasmid, the abundance of correctly initiated gamma-neo transcripts increased dramatically. However, beta-neo expression could not be activated by the LAR in K562 cells. These studies should allow us to further dissect the interactive roles of globin promoters and enhancers in K562 cells.
Ulrich
M J
MJ
Department of Medicine, Jewish Hospital, Washington University Medical Center, St. Louis, Missouri 63110.
Moon
A M
AM
Ley
T J
TJ
D002460:Cell Line; D015870:Gene Expression; D005809:Genes, Regulator; D005822:Genetic Vectors; D005914:Globins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014162:Transfection
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D005914:Globins